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Influence of carrageenan (e 407) on the membrane of enterocytes investigated by fluorescent probes.

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ПАТОФІЗІОЛОГІЯ
© Ye. O. Posokhov, A. S. Tkachenko*, Ye. M. Korniyenko**
UDC 547. 787. 2 + 535. 33/. 34
Ye. O. Posokhov, A. S. Tkachenko*, Ye. M. Korniyenko**
Influence of Carrageenan (E 407) on the Membrane of Enterocytes
Investigated by Fluorescent Probes Research Institute of Chemistry (Kharkiv)
*Kharkiv National Medical University (Kharkiv)
**V. N. Karazin Kharkiv National University (Kharkiv)
The results presented in this article were obtained
in following research «Investigation of long-term effects
of regular consumption of foods containing genetically
modified organisms in damaged epithelial barrier of the
gastrointestinal tract» (state registration number 0110
U000653).
Introduction. Food additive E 407 (carrageenan) is
widely used in the modern food industry. It is sulfated
polysaccharide, which is produced by extraction from
red seaweed. Carrageenan serves as natural thickener
in dairy, confectionery and meat products. Moreover, in
experimental medicine carrageenan is widely used to
model the inflammatory processes. It has been used in
the pathophysiology for modeling of peritonitis, pleurisy, arthritis and carrageenan-induced edema of the
limbs in rats [6, 13]. Carrageenan-induced inflammation, originally described by Winter, is an acute, nonimmune and easily reproducible. Edema and erythema,
hyperalgesia develop immediately after subcutaneous
injection of carrageenan and they are the result of proinflammatory agents such as bradykinin, histamine,
tachykinins, components of the complement system,
reactive oxygen and nitrogen species [6]. There is an
evidence of oncogenic transformation of cells under
the influence of carrageenan, the positive correlation
between the use of products with this additive in the
diet and an increased risk of breast carcinoma has
been found [17]. Also the model of ulcero-necrotizing
carrageenan-induced gastroenterocolitis has been reported [18]. In this connection, the question about the
safety of carrageenan as a food additive arises. In modern literature, there are no reliable data about the effect
of systematic consumption of carrageenan neither by
an adult nor by child or by pregnant women. A model
of moderate chronic carrageenan gastroenterocolitis
without necrotizing process was elaborated at Kharkiv
National Medical University on the basis of the model of
necrotizing carrageenan-induced gastroenterocolitis.
At the same time, the mechanism of carrageenan
action on enterocyte biomembranes during continious
consumption remains unexplored. It is very urgent task,
because of the spread of food products containing carrageenan in the diet of the consumers. The investigation
of the mechanisms of biomembranes damage will help
to develop regulation rules of carrageenan use in food.
The purpose of the research was to study the influence of carrageenan (E 407) on the lipid membrane
of enterocytes in rats with fluorescent probes (ortho-hydroxy derivatives of oxazole), which non-covalently bind
to cell membranes of experimental animals and have a
quick response to changes in their microenvironment
[3-5].
Subjects and methods. The female Wistar rats
were used for the experiment. Chronic carrageenaninduced gastroenterocolitis was reproduced by the
free access of animals to 1 % solution of carrageenan
in drinking water. Laboratory animals were divided into
2 groups. Group № 1 consisted of intact animals, group
№ 2 consisted of experimental animals with chronic
carrageenan-induced gastroenterocolitis. A month
after the start of the experiment animals were taken
from the experiment by decapitation. Manipulations
with animals were carried out in accordance with the
provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and
other Scientific Purposes (Strasbourg, 1986). The intestine was removed and placed on the cold immediately after decapitation of rats. Intestinal perfusion was
performed using cooled physiological solution. Epithelial cells were detached by scraping the inner surface
of the intestine by anatomical knife. A suspension of
epithelial cells was made in Tris-HCl buffer (pH 7. 4).
Fluorescent probes were dissolved in acetonitrile to
the initial concentration of 2?10?4 mol / l. 10 ?l of each
corresponding probe was added to the suspension of
enterocytes. The final concentration of each probe in
the suspensions of investigated membranes is 1?10?6
mol/l, thus, the molar ratio of lipid / probe was 1000:1.
Then, we measured the fluorescence probes O1O
(2 ? (2 ??OH-phenyl)-5-phenyl-1,3-oxazole) and PH7
(2 ? (2 ??OH-phenyl)-phenanthrene [10,11] ?1,3-oxazole) in physiological solutions containing enterocytes
of rats with chronic carrageenan-induced gastroenterocolitis. Measurement of the fluorescence was performed by spectrofluorometer «Hitachi F4010» after 1
hour after the addition of probes to a solution of cells.
The fluorescence spectra of probes were measured in
the diapason of 340-600 nm with monochromator slit
width of excitation and fluorescence 5 and 5 nm, respectively, and the excitation wavelength of 330 nm.
The enterocytes of intact healthy animals were used as
Вісник проблем біології і медицини ? 2013 ? Вип. 1, том 1 (98)
229
ПАТОФІЗІОЛОГІЯ
???????????? ??????????? ??????
? ???
T*
N
ESIPT
?????
O
P
O
O
O
N
O
P
O
O
O
O
O
375
375??
nm
-O
O
O
O
HO
N
O
330330
nm??
N
-O
N*
HO
+
+
Excited state
O
HO
N
O
O
470 ??
nm
470
O1O
N
HO
HO
N
O
T
HO
N
O
O
Ground
state ??????? ???
????????
???????????
N
PH7
Fig. 1. Scheme of excited state proton transfer (ESIPT)
reaction in 2 ? (2 ?-OH-phenyl)-5-phenyl-1,3-oxazole
(probe O1O). Upwards arrow shows the electronic
excitation and downwards arrow represents the light
emission (fluorescence). Corresponding maxima of
fluorescence are measured in nanometers.
Fig. 2. Expected location and orientation of fluorescent
probes O1O and PH7 based on their fluorescence properties in lipid membranes [3-5] and on the basis of their
structural similarity with fluorescent probes with known
localization in lipid membranes [15]. Two molecules
of phosphatidylcholine from the outer monolayer are
showed to denote the localization of the probes.
a control sample. The fluorescent probes, successfully
used in the past for studies of biological membranes
[3-5]: 2 ? (2 ?-OH-phenyl)-5-phenyl-1,3-oxazole (probe
O1O) and 2 ? (2 ?-OH-phenyl)-phenanthrene [10,11]
?1,3-oxazole (probe PH7), were used for investigation of carrageenan influence on the membrane of enterocytes. The choice of fluorescent probes O1O, PH7
(ortho-hydroxy 2,5-diaryl-1,3-oxazole) for investigation
of carrageenan influence on physico-chemical properties of biological membranes was due to the fact that
the fluorescent characteristics of the probes depend on
the physico-chemical properties of their microenvironment: hydrogen bonding ability (i. e., the ability to form
hydrogen bonds), the polarity and viscosity of the microenvironment [2-5, 8-10].
Results and discussion. It has been known that in
the excited state the ortho-hydroxy 2,5-diaryl-1,3-oxazole is characterized by excited state proton transfer
(ESIPT) reaction (Fig. 1): hydroxyl group in the orthoposition of the lateral benzene ring acts as protonodonor and the nitrogen atom of oxazole ring acts as proton
acceptor [2, 8-10]. The result of the ESIPT reaction is
the formation of phototautomeric form (T*), fluorescent
in significantly longer wavelengths in comparison with
the initial form (N*) [2, 8-10].
The presence of two-band fluorescence allows to
perform ratiometric measurement, i. e. to use the ratio
of phototautomeric form and the initial form fluorescence intensities (IT*/IN*) as a parameter for evaluation
of the physical and chemical properties of the microenvironment. The use of ratiometric fluorescent probes
allows to exclude the measurement error associated
with the deviation of the fluorescent probe concentration (e. g., uneven distribution of the fluorescent probe
in various membranes) and the measurement error associated with a deviation of fluorescence technique settings (deviation of the intensity of the exciting source, a
change in focus, changes in the sensitivity of the photodetector, etc.) [10].
Compounds that differ in their lipophilicity [3-5]
were selected for the present study. It is expected that
the regions of localization of selected probes in the
membrane are different and correspond to the lipophilicity of the probes (Fig. 2) [1, 3-5]. Expected location
and orientation of O1O and PH7 in lipid membranes is
based on their fluorescence properties in lipid membranes [3-5] and on the basis of their structural similarity with fluorescent probes with known localization in
lipid membranes [1]. The location of the probes in lipid
membrabes: probe O1O is located in the region of glycerol residues of phospholipids and in the region of carbonyl groups of phospholipids; probe PH7 is located in
the region of methylene chains of phospholipids and in
the center of the bilayer (Fig. 2).
A noticeable increase in the intensity and hypsochromic (i. e. short-wavelength) shift of the lonwavelength fluorescence band of probe O1O tautomeric
Table
Fluorescence intensity of probes O1O and PH7 in enterocyte membranes of animals with
carrageenan-induced gastroenterocolitis
Fluorescence intensity,a. u.
Sample
Control
Gastroenterocolitis
230
375 nm
5,0
5,3
Probe О1О
470 nm
23,7
79,4
I470/I375
4,7
15,0
425 nm
67,4
64,6
Probe РН7
485 nm
124,1
117,9
I485/I425
1,8
1,8
Вісник проблем біології і медицини ? 2013 ? Вип. 1, том 1 (98)
ПАТОФІЗІОЛОГІЯ
Entherocytes + ?407
Entherocytes
control
+ ?407
Fluorescence intensity, a.u.
80
60
40
20
0
350
400
4 50
500
550
600
650
Wavelength, nm
Fig. 3. The fluorescence spectra of the probe O1O in
solutions containing enterocytes: (a) control (solid line),
(b) the animals with carrageenan-induced gastroenterocolitis (dashed line).
form (IT*) was observed as a result of carrageenan action on enterocytes, at the same time, the intensity of
the fluorescence band of the probe O1O normal form
(IN*) was almost unchanged (Table, Fig. 3). Thus, the
intensity ratio of the tautomeric and the initial forms IT*/
IN* of probe O1O increases under the influence of carrageenan (E 407) (Fig. 4).
The increase of the intensity of the tautomeric form
fluorescence band (IT*), hypsochromic (i. e. short-wavelength) longwavelength shift of the tautomeric form
fluorescence band and increased ratio of IT*/IN* for O1O
probe indicates a decrease in polarity and hydrogenbonding ability of the microenvironment of probe O1O in
the enterocyte membranes of rats with gastroenterocolitis. Such decrease in polarity and hydrogen-bonding
ability of the microenvironment of probe O1O indicates
16
PROBE O1O, ENTHEROCYTES + E407
ENTHEROCYTES
I(470)/ I(400)
12
8
4
CONTROL
+ ?407
Fig. 4. The ratio of the tautomeric and the initial form
intensities IT*/IN* for O1O probe in the membranes
of enterocytes.
a decrease in hydration of the probe microenvironment in the erythrocyte membranes of the experimental
group.
Taking into consideration the emulsifying properties
of carrageenan [14, 16], the discussed reduction of hydration might be caused by the possible accumulation
of carrageenan (E 407) in the regions of probe O1O localization may increase the microviscosity of the lipid bilayer and, therefore, may lead to increase of orderliness
of the membrane phospholipid molecules (i. e. may
lead to the increase in membrane lipid order), which in
turn, may contribute in dehydration of the lipid bilayer
[7, 11, 12]. According to literature [7, 12], the similar
dehydration of the lipid membrane due to the increase
in orderliness of the phospholipid molecules (i. e. due
to the increase in membrane lipid order) is observed in
case of cholesterol and its esters accumulation in the
lipid bilayer. At the same time, in case of the probe pH
7 no significant change was found in its fluorescence
parameters under the influence of carrageenan (E 407)
on enterocytes (Table). The absence of changes in the
regions of the probe pH7 localization (i. e., in the rather
hydrophobic regions of the lipid bilayer) may be explained by the fact that, carrageenan, the structure of
which have polar hydroxyl and charged sulfonyl groups,
is localized in more polar regions of the lipid bilayer.
Thus, it was shown that fluorescent probe O1O (2 ?
(2 ?-OH-phenyl)-5-phenyl-1,3-oxazole) might be used
as an indicator for estimation of the nature and types
of changes in the structure of enterocyte membranes
of rats under the influence of carrageenan (E 407). It
was found that the changes in the membranes of enterocytes under the influence of carrageenan occurred
in the regions of probe O1O localization, i. e. in quite
polar regions of the membrane: presumably, in the region of glycerol residues of phospholipids and in the
region of the carbonyl groups of phospholipids. The influence of carrageenan (E 407) didn?t lead to changes
in the regions of probe pH 7 membrane localization, i. e.
in a more hydrophobic region of the enterocyte membranes: presumably, in the in the region of methylene
chains of the phospholipids and in the bilayer center.
It has been known that the absorption of essential
nutritional factors such as amino acids, glucose, vitamins takes place in the small intestine. Disturbance of
enterocyte membrane, which occurs under the influence of carrageenan, reduces the absorption of nutrients in the gastrointestinal tract, which may significantly
aggravate the course of gastroenterocolitis. In addition,
carrageenan-induced disorder of enterocyte transport
function may be explained by its possible effect on the
structure of protein transporters localized at the apical
surface of enterocytes, which indicates the impact of a
multi-vector influence of carrageenan on the morphofunctional state of gastrointestinal tarct in experimental
animals.
Conclusions.
1. The consumption of carrageenan leads to the following structural changes in the enterocyte membranes
of experimental animals: to increase in microviscosity of the lipid bilayer, to increase in the orderliness of
Вісник проблем біології і медицини ? 2013 ? Вип. 1, том 1 (98)
231
ПАТОФІЗІОЛОГІЯ
phospholipid molecules in the membrane (i. e. due to
the increase in membrane lipid order) and to dehydration of membranes.
2. The use of fluorescent probes (ortho-hydroxy
oxazole derivatives) allows to estimate the type and
nature of the damaging effect of carrageenan on the
structural components of membranes. Thus, the mentioned method, using ortho-hydroxy oxazole derivatives
as fluorescent probes, may be suggested for application in the practical medicine for the study of the negative effects on the human body similar to carrageenan
food supplements.
Prospects for further research. Our data substantiate the importance of research aimed at understanding the mechanisms of action of food additives
and to evaluate their safety for human health.
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УДК 547. 787. 2 + 535. 33/. 34
ДОСЛІДЖЕННЯ ВПЛИВУ КАРАГЕНІНУ (Е 407) НА МЕМБРАНИ ЕНТЕРОЦИТІВ МЕТОДОМ ФЛУОРЕСЦЕНТНИХ ЗОНДІВ
Посохов Є. О., Ткаченко А. С., Корніенко Є. М.
Резюме. За допомогою флуоресцентних зондів ? орто-гідроксипохідних оксазолу в експерименті на
щурах вивчено вплив на структури мембран ентероцитів тварин доданого до їх раціону каррагеніну (Е 407).
Встановлено, що каррагенін змінює гідратацію мембран ентероцитів експериментальних тварин. Показано,
що під впливом каррагеніну (Е 407) відбуваються зміни у полярних областях біомембран, в той же час,
вплив цієї харчової добавки не призводить до змін у гідрофобних ділянках мембран ентероцитів. Характер
виявлених змін дозволяє зробити висновок про збільшення мікров?язкості мембран ентероцитів щурів з
хронічним каррагенін-індуцірованнм гастроентероколітом.
Ключові слова: ентероцит, біомембрана, каррагенін, флуоресцентні зонди.
232
Вісник проблем біології і медицини ? 2013 ? Вип. 1, том 1 (98)
ПАТОФІЗІОЛОГІЯ
УДК 547. 787. 2 + 535. 33/. 34
ИССЛЕДОВАНИЕ ВЛИЯНИЯ КАРРАГЕНИНА (Е 407) НА МЕМБРАНЫ ЭНТЕРОЦИТОВ МЕТОДОМ
ФЛУОРЕСЦЕНТНЫХ ЗОНДОВ
Посохов Е. А., Ткаченко А. С., Корниенко Е. М.
Резюме. При помощи флуоресцентных зондов ? орто-гидроксипроизводных оксазола в эксперименте на крысах изучено влияние на структуры мембран энтероцитов животных добавленного в их рацион
каррагенина (Е 407). Установлено, что каррагенин изменяет гидратацию мембран энтероцитов экспериментальных животных. Показано, что под влиянием каррагенина (Е 407) происходят изменения в полярных областях биомембран, в то же время, воздействие этой пищевой добавки не приводит к изменениям
в гидрофобных участках мембран энтероцитов. Характер выявленных изменений позволяет сделать вывод
об увеличении микровязкости мембран энтероцитов крыс с хроническим каррагенин-индуцированнм
гастроэнтероколитом.
Ключевые слова: энтероциты, биомембрана, каррагенин, флуоресцентные зонды.
UDC 547. 787. 2 + 535. 33/. 34
Influence of Carrageenan (E 407) on the Membrane of Enterocytes Investigated by Fluorescent Probes
Posokhov Ye. O., Tkachenko A. S., Korniyenko Ye. M.
Summary. The influence on enterocytes membrane structure of added to the diet of rats food-additive carrageenan (E 407) was studied using fluorescent probes (derivatives of ortho-hydroxy oxazole). It was found that
carrageenan changed hydration membrane of enterocytes in experimental animals. It was shown that carrageenan
(E 407) caused changes in the polar regions of biological membranes and at the same time, the consumption of this
food-additive didn?t lead to any changes in the hydrophobic parts of the membrane of enterocytes. Nature of detected changes allows to make a conclusion that enterocyte membranes in rats with chronic carrageenan-induced
gastroenterocolitis have increased microviscosity.
Key words: enterocytes, biomembrane, carrageenan, fluorescent probes.
Стаття надійшла 15. 01. 2013 р.
Рецензент ? проф. Костенко В. О.
Вісник проблем біології і медицини ? 2013 ? Вип. 1, том 1 (98)
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fluorescence, enterocytes, investigated, carrageenan, 407, probes, membranes, influence
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